MerlinS13 phosphorylation regulates meningioma Wnt signaling and magnetic resonance imaging features.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with ß-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

Cilia-enriched oxysterol 7ß,27-DHC is required for polycystin ion channel activation.

Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7ß,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7ß,27-DHC-dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.

MerlinS13 phosphorylation controls meningioma Wnt signaling and magnetic resonance imaging features.

Meningiomas are the most common primary intracranial tumors and are associated with inactivation of the tumor suppressor NF2/Merlin, but one-third of meningiomas retain Merlin expression and typically have favorable clinical outcomes. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that predict meningioma outcomes and could be used to guide treatment de-escalation or imaging surveillance of Merlin-intact meningiomas are lacking. Here we integrate single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma cells, xenografts, and human patients to define biochemical mechanisms and an imaging biomarker that distinguish Merlin-intact meningiomas with favorable clinical outcomes from meningiomas with unfavorable clinical outcomes. We find Merlin drives meningioma Wnt signaling and tumor growth through a feed-forward mechanism that requires Merlin dephosphorylation on serine 13 (S13) to attenuate inhibitory interactions with ß-catenin and activate the Wnt pathway. Meningioma MRI analyses of xenografts and human patients show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC) on diffusion-weighted imaging. In sum, our results shed light on Merlin posttranslational modifications that regulate meningioma Wnt signaling and tumor growth in tumors without NF2/Merlin inactivation. To translate these findings to clinical practice, we establish a non-invasive imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with favorable meningiomas.

Ras-mutant cancers are sensitive to small molecule inhibition of V-type ATPases in mice.

Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

SOX11 Inhibitors Are Cytotoxic in Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is a fatal subtype of non-Hodgkin lymphoma. SOX11 transcription factor is overexpressed in the majority of nodal MCL. We have previously reported that B cell-specific overexpression of SOX11 promotes MCL pathogenesis via critically increasing BCR signaling in vivo. SOX11 is an attractive target for MCL therapy; however, no small-molecule inhibitor of SOX11 has been identified to date. Although transcription factors are generally considered undruggable, the ability of SOX11 to bind to the minor groove of DNA led us to hypothesize that there may exist cavities at the protein-DNA interface that are amenable to targeting by small molecules. EXPERIMENTAL DESIGN: Using a combination of in silico predictions and experimental validations, we report here the discovery of three structurally related compounds (SOX11i) that bind SOX11, perturb its interaction with DNA, and effect SOX11-specific anti-MCL cytotoxicity. RESULTS: We find mechanistic validation of on-target activity of these SOX11i in the inhibition of BCR signaling and the transcriptional modulation of SOX11 target genes, specifically, in SOX11-expressing MCL cells. One of the three SOX11i exhibits relatively superior in vitro activity and displays cytotoxic synergy with ibrutinib in SOX11-expressing MCL cells. Importantly, this SOX11i induces cytotoxicity specifically in SOX11-positive ibrutinib-resistant MCL patient samples and inhibits Bruton tyrosine kinase phosphorylation in a xenograft mouse model derived from one of these subjects. CONCLUSIONS: Taken together, our results provide a foundation for therapeutically targeting SOX11 in MCL by a novel class of small molecules.

A systems-biology approach to molecular machines: Exploration of alternative transporter mechanisms.

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.

A kinetic mechanism for enhanced selectivity of membrane transport.

Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this 'slip' mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a 'detoxification' mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development.

A Triazolotriazine-Based Dual GSK-3ß/CK-1δ Ligand as a Potential Neuroprotective Agent Presenting Two Different Mechanisms of Enzymatic Inhibition.

Glycogen synthase kinase 3ß (GSK-3ß) and casein kinase 1δ (CK-1δ) are emerging targets for the treatment of neuroinflammatory disorders, including Parkinson's disease. An inhibitor able to target these two kinases was developed by docking-based design. Compound 12, 3-(7-amino-5-(cyclohexylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)-2-cyanoacrylamide, showed combined inhibitory activity against GSK-3ß and CK-1δ [IC50 (GSK-3ß)=0.17 µm; IC50 (CK-1δ)=0.68 µm]. In particular, classical ATP competition was observed against CK-1δ, and a co-crystal of compound 12 inside GSK-3ß confirmed a covalent interaction between the cyanoacrylamide warhead and Cys199, which could help in the development of more potent covalent inhibitors of GSK-3ß. Preliminary studies on in vitro models of Parkinson's disease revealed that compound 12 is not cytotoxic and shows neuroprotective activity. These results encourage further investigations to validate GSK-3ß/CK-1δ inhibition as a possible new strategy to treat neuroinflammatory/degenerative diseases.

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

Cilia-Associated Oxysterols Activate Smoothened.

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11ß2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.

Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site.

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.

Stochastic steps in secondary active sugar transport.

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake.

Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.

Development and Application of a Virtual Screening Protocol for the Identification of Multitarget Fragments.

In this study, we report on a virtual ligand screening protocol optimized to identify fragments endowed with activity at multiple targets. Thanks to this protocol, we were able to identify a fragment that displays activity in the low-micromolar range at both ß-secretase 1 (BACE-1) and glycogen synthase kinase 3ß (GSK-3ß). These two structurally and physiologically unrelated enzymes likely contribute, through different pathways, to the onset of Alzheimer's disease (AD). Therefore, their simultaneous inhibition holds great potential in exerting a profound effect on AD. In perspective, the strategy outlined herein can be adapted to other target combinations.

Versatility of the Curcumin Scaffold: Discovery of Potent and Balanced Dual BACE-1 and GSK-3ß Inhibitors.

The multitarget approach has gained increasing acceptance as a useful tool to address complex and multifactorial maladies such as Alzheimer's disease (AD). The concurrent inhibition of the validated AD targets ß-secretase (BACE-1) and glycogen synthase kinase-3ß (GSK-3ß) by attacking both ß-amyloid and tau protein cascades has been identified as a promising AD therapeutic strategy. In our study, curcumin was identified as a lead compound for the simultaneous inhibition of both targets; therefore, synthetic efforts were dedicated to obtaining a small library of novel curcumin-based analogues, and a number of potent and balanced dual-target inhibitors were obtained. In particular, 2, 6, and 7 emerged as promising drug candidates endowed with neuroprotective potential and brain permeability. Notably, for some new compounds the symmetrical diketo and the ß-keto-enol tautomeric forms were purposely isolated and tested in vitro, allowing us to gain insight into the key requirements for BACE-1 and GSK-3ß inhibition.

Ligand-Based Discovery of a New Scaffold for Allosteric Modulation of the µ-Opioid Receptor.

With the hope of discovering effective analgesics with fewer side effects, attention has recently shifted to allosteric modulators of the opioid receptors. In the past two years, the first chemotypes of positive or silent allosteric modulators (PAMs or SAMs, respectively) of µ- and δ-opioid receptor types have been reported in the literature. During a structure-guided lead optimization campaign with µ-PAMs BMS-986121 and BMS-986122 as starting compounds, we discovered a new chemotype that was confirmed to display µ-PAM or µ-SAM activity depending on the specific substitutions as assessed by endomorphin-1-stimulated ß-arrestin2 recruitment assays in Chinese Hamster Ovary (CHO)-µ PathHunter cells. The most active µ-PAM of this series was analyzed further in competition binding and G-protein activation assays to understand its effects on ligand binding and to investigate the nature of its probe dependence.

Multitarget drug discovery for Alzheimer's disease: triazinones as BACE-1 and GSK-3ß inhibitors.

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3ß. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03±0.01) µM and (14.67±0.78) µM for BACE-1 and GSK-3ß, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.